ADP-dependent glucokinase controls metabolic fitness in prostate cancer progression

Background Cell metabolism plays a pivotal role in tumor progression,and targeting cancer metabolism might effectively kill cancer cells.We aimed to investigate the role of hexokinases in prostate cancer(PCa)and identify a crucial target for PCa treatment.Methods The Cancer Genome Atlas(TCGA)database,online tools and clinical samples were used to assess the expression and prognostic role of ADP-dependent glucokinase(ADPGK)in PCa.The effect of ADPGK expression on PCa cell malignant phenotypes was validated in vitro and in vivo.Quantitative proteomics,metabolomics,and extracellular acidification rate(ECAR)and oxygen consumption rate(OCR)tests were performed to evaluate the impact of ADPGK on PCa metabolism.The underlying mechanisms were explored through ADPGK overexpression and knockdown,co-immunoprecipitation(Co-IP),ECAR analysis and cell counting kit-8(CCK-8)assays.Results ADPGK was the only glucokinase that was both upregulated and predicted worse overall survival(OS)in prostate adenocarcinoma(PRAD).Clinical sample analysis demonstrated that ADPGK was markedly upregulated in PCa tissues vs.non-PCa tissues.High ADPGK expression indicates worse survival outcomes,and ADPGK serves as an independent factor of biochemical recurrence.In vitro and in vivo experiments showed that ADPGK overexpression promoted PCa cell proliferation and migration,and ADPGK inhibition suppressed malignant phenotypes.Metabolomics,proteomics,and ECAR and OCR tests revealed that ADPGK significantly accelerated glycolysis in PCa.Mechanistically,ADPGK binds aldolase C(ALDOC)to promote glycolysis via AMP-activated protein kinase(AMPK)phosphorylation.ALDOC was positively correlated with ADPGK,and high ALDOC expression was associated with worse survival outcomes in PCa.Conclusions In summary,ADPGK is a driving factor in PCa progression,and its high expression contributes to a poor prognosis in PCa patients.ADPGK accelerates PCa glycolysis and progression by activating ALDOC-AMPK signaling,suggesting that ADPGK might be an effective target and marker for PCa treatment and prognosis evaluation.

Glucokinase regulatory protein rs780094 polymorphism is associated with type 2 diabetes mellitus, dyslipidemia, non-alcoholic fatty liver disease, and nephropathy

In this editorial,we comment on the article by Liu et al published in the recent issue of the World Journal of Diabetes(Relationship between GCKR gene rs780094 polymorphism and type 2 diabetes with albuminuria).Type 2 diabetes mellitus(T2DM)is a chronic disorder characterized by dysregulated glucose homeostasis.The persistent elevated blood glucose level in T2DM significantly increases the risk of developing severe complications,including cardiovascular disease,re-tinopathy,neuropathy,and nephropathy.T2DM arises from a complex interplay between genetic,epigenetic,and environmental factors.Global genomic studies have identified numerous genetic variations associated with an increased risk of T2DM.Specifically,variations within the glucokinase regulatory protein(GCKR)gene have been linked to heightened susceptibility to T2DM and its associated complications.The clinical trial by Liu et al further elucidates the role of the GCKR rs780094 polymorphism in T2DM and nephropathy development.Their findings demonstrate that individuals carrying the CT or TT genotype at the GCKR rs780094 locus are at a higher risk of developing T2DM with albuminuria compared to those with the CC genotype.These findings highlight the importance of genetic testing and risk assessment in T2DM to develop effective preventive strategies and personalized treatment plans.

Facilitating effects of berberine on rat pancreatic islets through modulating hepatic nuclear factor 4 alpha expression and glucokinase activity

AIM： To observe the effect of berberine on insulin secretion in rat pancreatic islets and to explore its possible molecular mechanism. METHODS： Pdmary rat islets were isolated from male Sprague-Dawley rats by collagenase digestion and treated with different concentrations （1, 3, 10 and 30 μmol/L） of berberine or 1 μmol/L Glibenclamide （GB） for 24 h. Glucose-stimulated insulin secretion （GSIS） assay was conducted and insulin was determined by radioimmunoassay. 3-（4,5-Dimethylthiazol-2-yl）- 2,5-diphenyltetrazolium bromide （MTT） assay was performed to evaluate cytotoxicity. The mRNA level of hepatic nuclear factor 4 alpha （HAIF4α） was determined by reverse transcription polymerase chain reaction （RT-PCR）. Indirect immunofluorescence staining and Western blot analysis were employed to detect protein expression of HNF4α in the islets. Glucokinase （GK） activity was measured by spectrophotometric method. RESULTS： Berberine enhanced GSIS rather than basal insulin secretion dose-dependently in rat islets and showed no significant cytotoxicity on islet cells at the concentration of 10 μmol/L. Both mRNA and protein expressions of HNF4α were up-regulated by berberine in a dose-dependent manner, and GK activity was also increased accordingly. However, GB demonstrated no regulatory effects on HNF4α expression or GK activity. CONCLUSION： Berberine can enhance GSIS in rat islets, and probably exerts the insulinotropic effect via a pathway involving HNF4α and GK, which is distinct from sulphonylureas （SUs）.

Establishment of Hypoglycemic Agent Screening Method Based on Human Glucokinase

Objective To establish a reliable platform for screening glucokinase activators （GKAs） in vitro. Methods Pancreatic glucokinase （PGK） protein expressed in a prokaryotic expression system as a histidine-tagged fusion protein from Homo sapiens was produced. Then, response surface methodology （RSM） was used to optimize the microplate-based GKA screening platform. In the f＇trst step of optimization with Plackett-Burman design （PBD）, initial pH, reaction time and MgC12 were found to be important factors affecting the activity ratio of GKA （RO-28-1675） significantly. In the second step, a 23 full factorial central composite design （CCD） and RSM were applied to the optimal condition determination of each significant variable. A second-order polynomial was determined by a multiple regression analysis of the experimental data. Results The following optimal values for the critical factors were obtained： initial pH 0 （7.0）, reaction time-0.63 （13.7 min） and MgC12 0.11 （2.11 mmol/L） with a predicted value of the maximum activity ratio of 34.1%. Conclusion Under the optimal conditions, the practical activity ratio is 34.8%. The determination coefficient （R2） is 0.9442, ensuring adequate credibility of the model. LLAE3, extracted from Folium nelumbinis in our laboratory, has prominently activated effects on PGK.

Seropositivity rates of water channel protein 4 antibodies compared between a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay in neuromyelitis optica patients

A total of 66 samples （from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, aa 13 cases with optic neuritis） were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay. The sensitivities and specificities of the two assays were similar. We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay. A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the se of neuromyelitis optica patients. No significant correlations were identified with onset age or disea duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica. The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.

Effects of vanadate on the activities of mice glucokinase and hexokinase

This study aimed at acquiring knowledge on the hypoglycemic mechanisms of sodium metavanadate (SMV) showed that the liver glucokinase and muscle hexokinase activities increased rapidly after oral SMV was given, and that the blood glucose level was correlated closely with the activities of the two enzymes but not with the insulin level; which indicated that SMV could improve the altered glucose phosphorylation in diabetic mice independently of stimulating insulin secretion. This was probably one of the mechanisms of hypoglycemic effects of SMV.

Determination of anti-endomysium IgA antibodies in the diagnosis of celiac disease:Comparison of a novel ELISA-based assay with conventional immunofluorescence

AIM： To evaluate the novel anti-endomysium （anti-EMA） detection based on ELISA. METHODS： Anti-EMA IgA was measured by a novel ELISA in 196 patients with gastrointestinal symptoms and suspected mal-absorption. Data were compared with those obtained by the conventional IF test. RESULTS： A good concordance of 98% was found between these two assays. In sera of 161 patients （82%） both assays tested negative whereas in sera of 31 patients （16%） both assays tested positive for the presence of anti-EMA antibodies. Discrepancies between EMA- ELISA and EMA-immunofluorescence （IF） were found in only 4 patients （2%）.CONCLUSION： This ELISA can replace IF for the detection of anti-EMA antibodies and provide clinicians with an excellent tool to screen for celiac disease in patients with gastrointestinal complaints.

Immunofluorescence on paraffin embedded renal biopsies:Experience of a tertiary care center with review of literature

AIMTo describe the technique of immunofluorescence on paraffin embedded tissue sections and discuss the po-tential pitfalls with an in depth review of literature.METHODSImmunofluorescence is integral to diagnostic renal pa-thology. Immunofluorescence on paraffin embedded renal biopsies （IF-P） after enzyme treatment has been described in literature, however has not found widespread use in renal pathology laboratories. In our laboratory proteinase K digestion of paraffn embedded renal biopsy material was standardized and applied prospectively in cases where immunofuorescence on fresh frozen tissue was non contributory or not possible. Diagnostic utility was assessed and in a cohort of cases comparison of intensity of staining with routine immunofuorescence was performed.RESULTSOver the 5-year study period, of the 3141 renal biopsies received IF-P was performed on 246 cases （7.7%） and was interpretable with optimal digestion in 214 cases （6.8%）. It was of diagnostic utility in the majority of cases, which predominantly included glomerular disease. Non-diagnostic IF-P was found in membranous nephropathy （2 of 11 cases）, membranoproliferative glomerulonephritis （2 of 32 cases）, lupus nephritis （1 of 25 cases）, post infectious glomerulonephritis （1 of 11 cases） and chronic glomerulonephritis （3 of 8 cases）. Comparing cases with both routine IF and IF-P, 35 of 37 showed either equal intensity or a minor difference in intensity of staining（1＋） for the diagnostic immunoglobulin/complement. Technically assessment of immunofluorescence on the paraffin embedded tissue was found to be easier with clearly observed morphology, however a false positive staining pattern was observed in under-digested tissue. CONCLUSIONAs a “salvage” technique, immunofuorescence on paraffn embedded renal biopsies is of great diagnostic utility, however not without pitfalls.

Sapium ellipticum(Hochst) Pax ethanol leaf extract modulates glucokinase and glucose-6-phosphatase activities in streptozotocin induced diabetic rats

Objective:To examine the effects of Sapium ellipticum(SE) leaf extract on the hepatic activities of glucokinase and glucose-6-phosphatase in streptozotocin-induced diabetic Wistar rats.Methods:STZ-induced diabetic Wistar rats(four groups,n = 8) were used in this study.SE was assessed at two different doses,400 and 800 mg/kg BW,in comparison with metformin(METF)(12 mg/kg BW) as a reference antidiabetic drug.All treatments were done orally(p.o),twice daily at 8 h interval for a period of 21 days.Glucokinase and glucose-6-phosphatase activities were respectively determined using standard protocols.Hepatic and muscle glycogen contents were estimated as well.Results:STZ caused significant decrease in glucose-6-phosphatase activity and concomitant increase in glucokinase activity.SE extract especially at 400 mg dosage significantly reversed the alterations by increasing glucokinase activity by 40.31% and inhibiting glucose-6-phosphatase activity by 37.29% compared to diabetic control animals.However,the effects were significantly lower than that of METF which enhanced glucokinase activity by94.76% and simultaneously inhibited glucose-6-phosphatase activity by 49.15%.The extract also improved hepatic glycogen level by 32.37 and 27.06% at 400 and 800 mg dosage respectively.HPLC-MS analysis of some SE fractions in dynamic MRM mode(using the optimized compound-specific parameters) revealed among other active compounds,the presence of amentoflavone,which has been associated with antidiabetic function.Conclusions:The ability of SE extract to concurrently inhibit glucose-6-phosphatase and activate glucokinase in this study suggests that it may be a treatment option for type 2 diabetes patients,and the presence of amentoflavone in the plant extract may account for its anti-diabetic potential.

Persian Shallot (<i>Allium hirtifolium</i>Boiss) Extract Elevates Glucokinase (GCK) Activity and Gene Expression in Diabetic Rats

Hepatic GCK is a key enzyme in glucose homeostasis and, as such, is a potential target for treatment strategies of diabetes. We investigated the effect of Persian shallot (Allium hirtifolium Boiss) hydroalchoholic extract on blood glucose level, plasma insulin level, GCK activity and its gene expression. Thirty two male rats were divided into 4 groups of 8, diabetic groups received 100 and 200 mg/kg Persian shallot extract, diabetic control and normal control received 0.9% saline for 30 days. Investigations of gene expression by Real-Time PCR showed that Persian shallot had led to gently increased GCK gene expression in diabetic rats. GCK activity increased significantly in Persian shallot treated group in dose dependent manner (P < 0.05). These results indicated that Persian shallot exhibited a significant potential as a hypoglycemic agent perhaps via its ability to enhance insulin secretion, GCK gene expression and its activity.

Analysis of the glucokinase gene in Iranian families with maturity onset diabetes of the young

Non insulin dependent diabetes mellitus (NIDDM) as a most common form of diabetes is a major public health problem;there is a subgroup of NIDDM patients who develop the disease at an early age and show a dominant mode of inheritance. This type is nominates Maturity onset diabetes of the young (MODY). The prevalence of MODY is difficult to access, and patients with MODY genes mutations are often identified during routine screening for other purposes. MODY2 was linked to glucokinase gene (GCK) mutations, and accounted for 8% to 56% of MODY, with the highest prevalence found in the southern Europe. The aim of this study was to examine the prevalence and nature of mutations in GCK gene in Iranian paients. We have screened GCK mutations by polymerase chain reaction (PCR);single stranded conformation polymorphism (SSCP) technique in 12 Iranian families with clinical diagnosis of MODY, included 30 patients (8 males and 22 females) and their 21 family members. PCR products with abnormal mobility in denaturing gradient gel electrophoresis (DGGE) were directly sequenced. We identified 6 novel mutations in GCK gene in Iranian families (corresponding to 36.6% prevalence). Our findings and the last study on MODY1 highlight that in addition to GCK, other MODY genes such as MODY3 and MODYX may play a significant role in diabetes characterized by monogenic autosomal dominant transmission. There is an important point that the genetic recognation can be used to pre-symptomatically identify family members at risk for developing MODY.

COMPARISON BETWEEN IMMUNOFLUORESCENCE AND PCR IN DETECTING HUMAN PAPILLOMA VIRUS IN CONDYLOMA ACUMINATA

Objective To compare the effectiveness of immunofluorescence and polymerase chain reaction （PCR） in detecring human papilloma virus （HPV） in condyloma acuminata （CA）. Methods HPVs in CA tissues from 60 patients were detected by immunofluorescence and PCR, respectively. Different subtypes of HPVs were also identified with restriction fragment length polymorphism （RFLP） . Resuits The positive detective rates of immunofluorescence and PCR were 56. 67 % （34/60） and 96. 67 % （58/ 60）, respectively （ P 〈 0. 01 ）. RFLP results showed HPV6 and HPV11 were the main subtypes in the detected virus, which accounted for 98.28%. Conclusion The sensibility of PCR is superior to that of immunofluorescence.

The Application of Quantum Dots Double-Labeling Immunofluorescence Technology in Detection of PR and CD146 in Paraffin-Embedded Tissue Sections of Endometrioid Adenocarcinoma

Objective: The aim was to detect the expression of PR and CD146 in paraf-fin-embedded tissue sections of endometrioid adenocarcinoma by using QDs double-labeling immunofluorescence, and evaluate the applied value of QDs double-labeling immunofluorescence in endometrioid adenocarcinoma. Methods: To detect the expression of PR and CD146 on 140 cases of paraffin-embedded tissue sections of endometrioid adenocarcinoma by using QDS double-labeling immunofluorescence. Results: The co-expression of PR and CD146 in the endometrioid adenocarcinoma can be detected by QDs double-labeling immunofluorescence, and there was no correlation between them (P > 0.05). Conclusion: QDs double-labeling immunofluorescence can detect the localization and co-expression of PR and CD146 in the endometrioid adenocarcinoma.

Comparison of indirect immunofluorescence and western blot method in the diagnosis of hantavirus infections

BACKGROUND Serologic cross-reactivity between hantaviruses often complicates the interpretation of the results.AIM To analyze the diagnostic value of indirect immunofluorescence assay(IFA)and western blot(WB)in the diagnosis of hantavirus infections.METHODS One hundred eighty-eight serum samples from Puumala(PUUV)and Dobrava(DOBV)orthohantavirus infected patients were analyzed.Serology was performed using commercial tests(Euroimmun,Lübeck,Germany).RESULTS Using IFA,49.5%of acute-phase samples showed a monotypic response to PUUV,while 50.5% cross-reacted with other hantaviruses.The overall cross-reactivity was higher for immunoglobulin G(IgG)(50.0%)than for immunoglobulin M(IgM)(25.5%).PUUV IgM/IgG antibodies showed low/moderate reactivity with orthohantaviruses Hantaan(12.3%/31.5%),Seoul(7.5%/17.8%),DOBV(5.4%/28.1%),and Saaremaa(4.8%/15.7%).Both DOBV IgM and IgG antibodies were broadly reactive with Hantaan(76.2%/95.2%),Saaremaa(80.9%/83.3%),and Seoul(78.6%/85.7%)and moderate with PUUV(28.5%/38.1%).Using a WB,serotyping was successful in most cross-reactive samples(89.5%).CONCLUSION The presented results indicate that WB is more specific than IFA in the diagnosis of hantavirus infections,confirming serotype in most IFA cross-reactive samples.

New highly specific anti-BnASY polyclonal antibody prepara-tion and application in immunofluorescence assay

ASY （asynaptic） is a synaptonemal complex （SC） related protein in plant mei- osis. Some ASYs in plants have been cloned and functional determined. ASY in Brassica napus has not been sequenced and functional certificated. Here, we first cloned ASY gene in Brassica napus （BnASY） and inserted it into vector pET-32a for expression fusion protein BnASY-6His in Escherichia coli BL21 （DE3）. Purified fusion protein was used to produce polyclonal antibody. Specificity and application of polyclonal antibody was examined by Western blot and immunofluorescence assay. Results showed that BnASY-6His recombi-nant protein and anti-BnASY antibody were successfully obtained. Polyclonal anti-BnASY antibody had high specificity. Results of immunofluorescence showed that ASY signals appeared at leptotene stage, faded away gradually at pachytene stage and almost disap-peared at diplonema stage. In this study, highly specific polyclonal antibody was success-fully acquired and used for immunofluorescence assay in plant cells. It will contribute to functional research of ASY in B. napus.We thank Zhigang Li for providing pET-32a vector and Escherichia coli BL21 pLysS strains.

Allosteric process of human glucokinase conducive to fight against diabetes

More than 200 million people worldwide have diabetes. In China alone, about 60 million people

真菌病毒Beauveria bassiana chrysovirus 2(BbCV2)外壳蛋白多克隆抗体制备及应用

球孢白僵菌Beauveriabassiana是一种在害虫生物防治中应用广泛的虫生真菌,真菌病毒能够影响球孢白僵菌的致病力和生物学性状,但真菌病毒相关蛋白的抗体制备和利用其进行病毒检测和定位鲜有报道。本研究原核表达了一株普遍流行并且造成寄主球孢白僵菌毒力下降的双链RNA(double stranded RNA,dsRNA)病毒Beauveria bassiana chrysovirus 2(Bb CV2)的外壳蛋白(Coat protein,CP),并制备了多克隆抗体,进行了病毒在寄主球孢白僵菌胞内和胞外的检测,以及利用间接免疫荧光进行病毒在寄主内的定位。结果表明,表达的BbCV2-CP蛋白主要以包涵体的形式存在,相对分子质量约为85.7kD;经间接ELISA方法测定,制备的BbCV2-CP兔源多抗效价达到1:8192000;Westernblot结果显示,制备的BbCV2兔源多抗能与抗原蛋白进行特异性结合,且能检测出感染寄主球孢白僵菌的BbCV2;ELISA测定结果表明病毒BbCV2可在寄主真菌体内复制,并能够游离到寄主体外;经间接免疫荧光观察,发现BbCV2病毒定位于真菌细胞核中。本研究建立了球孢白僵菌真菌病毒的血清学检测体系,为球孢白僵菌-真菌病毒互作机制研究提供材料。

果胶酶对灵武长枣果实发育中阿拉伯半乳糖蛋白分布的影响

以3种不同浓度的果胶酶处理灵武长枣(Ziziphus jujuba‘Lingwu Changzao’)4个不同发育时期的果实,采用免疫细胞化学技术,在细胞水平上对果实阿拉伯半乳糖蛋白(AGPs)表位进行原位分析,探讨果胶酶对其不同发育时期的果实中AGPs分布的影响,为明确果胶酶对果实成熟软化的影响提供解剖学依据。结果表明:JIM13、JIM8和MAC204抗体所标记的抗原荧光强弱在各时期果实的不同组织存在一定的差异。当果肉组织用较低浓度的果胶酶0.028 U·mL^(-1)(E1)处理,果皮组织结构没有明显的变化,果皮及内部薄壁细胞细胞壁表面和细胞间隙中的AGPs抗原表位减少;0.056 U·mL^(-1)(E2)和0.084 U·mL^(-1)(E3)浓度果胶酶处理的果实,果实组织细胞壁解体程度增加,果皮及内部薄壁细胞细胞壁中AGPs抗原表位检测量逐渐降低,果胶酶浓度的增加导致AGPs在果实所有表位排列的更大影响和较低的荧光信号。经果胶酶最高浓度0.084 U·mL^(-1)(E3)处理后,Calcofluor White染色显示细胞壁区域荧光也不同程度减弱或降低,AGPs分布的紊乱和抗原表位的缺失与细胞中纤维素组装的变化有关。比较分析表明,不同发育时期的果实AGPs碳水化合物的分布不同,这与组织结构的变化有关;果胶酶作用下AGPs聚糖链的缺失导致细胞壁组分之间的相关性建立和细胞壁结构的重塑受阻,诱导了整个细胞壁结构的改变,影响了果实的成熟。

多重微球流式免疫荧光技术对肺癌患者外周血IL-1β、IL-6、IFN-γ水平测定及与hs-CRP的相关性分析

目的探讨多重微球流式免疫荧光技术在肺癌患者外周血白细胞介素(IL)-1β、IL-6和干扰素γ(IFN-γ)细胞因子的表达情况及与超敏C反应蛋白(hs-CRP)相关性分析。方法采用回顾性研究的方法,选择张家港市中医医院(下称该院)肿瘤科和肺病科收治的30例肺癌患者作为肺癌组,另选择同期该院收治的30例肺炎患者作为肺炎组,同时选取同期该院体检健康者30例作为对照组,通过多重微球流式免疫荧光技术检测血清中IL-1β、IL-6和IFN-γ水平,同时利用乳胶增强免疫散射比浊法测定外周血全血中hs-CRP水平。结果肺癌组与对照组比较,IL-1β、IL-6和hs-CRP水平显著升高,差异有统计学意义(P<0.001),而IFN-γ水平差异无统计学意义(P>0.05);肺炎组与对照组比较,IL-1β、IL-6、IFN-γ、hs-CRP水平均显著升高,差异有统计学意义(P<0.001);肺癌组与肺炎组比较,IL-6、IFN-γ、hs-CRP水平明显降低且IL-1β水平升高,差异均有统计学意义(P<0.05);肺癌组、肺炎组IL-6和hs-CRP水平分别进行Pearson相关性分析,均无明显的相关性。结论IL-1β、IL-6、IFN-γ可以作为有效评估肺癌患者免疫功能的辅助指标,多重微球流式免疫荧光技术的应用对于肺癌的诊断及监测具有潜在的临床应用价值。

鸡马立克氏病火鸡疱疹病毒间接免疫荧光检测方法的建立与比较

为建立鸡马立克氏病火鸡疱疹病毒(MDV HVT)的间接免疫荧光方法,将MDV HVT病毒接种96孔细胞板培养(CEF),72 h后用鸡抗马立克氏病病毒阳性血清作为一抗和兔抗鸡荧光抗体作为二抗,对反应条件进行优化,建立了间接免疫荧光方法(IFA)。并与经典的马立克蚀斑计数方法进行病毒滴度的测定比较,运用统计学方法分析,P小于0.05,相关系数为0.995,说明两种方法相关性很好,两种方法均可用于马立克氏病病毒的滴度测定。IFA方法较蚀斑计数方法操作简便快捷,可代替蚀斑计数方法用于马立克氏病活疫苗的病毒含量的初步测定,同时为2025年版《中国兽药典》马立克氏病活疫苗增加IFA鉴别检验方法积累实验数据。